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- EMDB-52311: Cryo-EM structure of the glucose-specific PTS transporter IICB fr... -

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Basic information

Entry
Database: EMDB / ID: EMD-52311
TitleCryo-EM structure of the glucose-specific PTS transporter IICB from E. coli in an intermediate state
Map data
Sample
  • Complex: homo-dimeric complex
    • Protein or peptide: PTS system glucose-specific EIICB component
  • Ligand: DODECYL-BETA-D-MALTOSIDE
Keywordsglucose transport protein / intermediate state / stalling / membrane protein / TRANSPORT PROTEIN
Function / homology
Function and homology information


protein-phosphocysteine-glucose phosphotransferase system transporter activity / protein-Npi-phosphohistidine-D-glucose phosphotransferase / protein-N(PI)-phosphohistidine-sugar phosphotransferase activity / D-glucose import across plasma membrane / D-glucose transmembrane transporter activity / D-glucose transmembrane transport / phosphoenolpyruvate-dependent sugar phosphotransferase system / transmembrane transporter complex / kinase activity / regulation of DNA-templated transcription ...protein-phosphocysteine-glucose phosphotransferase system transporter activity / protein-Npi-phosphohistidine-D-glucose phosphotransferase / protein-N(PI)-phosphohistidine-sugar phosphotransferase activity / D-glucose import across plasma membrane / D-glucose transmembrane transporter activity / D-glucose transmembrane transport / phosphoenolpyruvate-dependent sugar phosphotransferase system / transmembrane transporter complex / kinase activity / regulation of DNA-templated transcription / membrane / plasma membrane
Similarity search - Function
Phosphotransferase system, maltose/glucose-specific subfamily IIC component / PTS system glucose-specific IIBC component / : / Phosphotransferase system, IIB component, type 1 / Phosphotransferase system, EIIC component, type 1 / Phosphotransferase system EIIB, cysteine phosphorylation site / Glucose permease domain IIB / phosphotransferase system, EIIB / PTS EIIB domains cysteine phosphorylation site signature. / PTS_EIIB type-1 domain profile. ...Phosphotransferase system, maltose/glucose-specific subfamily IIC component / PTS system glucose-specific IIBC component / : / Phosphotransferase system, IIB component, type 1 / Phosphotransferase system, EIIC component, type 1 / Phosphotransferase system EIIB, cysteine phosphorylation site / Glucose permease domain IIB / phosphotransferase system, EIIB / PTS EIIB domains cysteine phosphorylation site signature. / PTS_EIIB type-1 domain profile. / PTS_EIIC type-1 domain profile. / Phosphotransferase system, EIIC / Phosphotransferase system, EIIC
Similarity search - Domain/homology
PTS system glucose-specific EIICB component
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.53 Å
AuthorsRoth P / Fotiadis D
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation184980 Switzerland
CitationJournal: J Struct Biol X / Year: 2025
Title: Cryo-EM structure of a phosphotransferase system glucose transporter stalled in an intermediate conformation.
Authors: Patrick Roth / Dimitrios Fotiadis /
Abstract: The phosphotransferase system glucose-specific transporter IICB serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IIC domain and ...The phosphotransferase system glucose-specific transporter IICB serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IIC domain and phosphorylates the substrate within the cell to produce the glycolytic intermediate, glucose-6-phosphate, through the IIB domain. Furthermore, IIC consists of a transport (TD) and a scaffold domain, with the latter being involved in dimer formation. Transport is mediated by an elevator-type mechanism within the IIC domain, where the substrate binds to the mobile TD. This domain undergoes a large-scale rigid-body movement relative to the static scaffold domain, translocating glucose across the membrane. Structures of elevator-type transporters are typically captured in either inward- or outward-facing conformations. Intermediate states remain elusive, awaiting structural determination and mechanistic interpretation. Here, we present a single-particle cryo-EM structure of purified, -dodecyl-β-D-maltopyranoside-solubilized IICB from . While the IIB protein domain is flexible remaining unresolved, the dimeric IIC transporter is found trapped in a hitherto unobserved intermediate conformational state. Specifically, the TD is located halfway between inward- and outward-facing states. Structural analysis revealed a specific -dodecyl-β-D-maltopyranoside molecule bound to the glucose binding site. The sliding of the TD is potentially impeded halfway due to the bulky nature of the ligand and a shift of the thin gate, thereby stalling the transporter. In conclusion, this study presents a novel conformational state of IIC, and provides new structural and mechanistic insights into a potential stalling mechanism, paving the way for the rational design of transport inhibitors targeting this critical bacterial metabolic process.
History
DepositionDec 11, 2024-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52311.png

Downloads & links

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Map

FileDownload / File: emd_52311.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 260 pix.
= 262.8 Å		1.01 Å/pix.
x 260 pix.
= 262.8 Å		1.01 Å/pix.
x 260 pix.
= 262.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.01077 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.053168107 - 1.6334897
Average (Standard dev.)0.0008439163 (±0.017799933)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 262.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52311_msk_1.map
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Half map: #2

Fileemd_52311_half_map_1.map
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Half map: #1

Fileemd_52311_half_map_2.map
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Sample components

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Entire : homo-dimeric complex

EntireName: homo-dimeric complex
Components
  • Complex: homo-dimeric complex
    • Protein or peptide: PTS system glucose-specific EIICB component
  • Ligand: DODECYL-BETA-D-MALTOSIDE

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Supramolecule #1: homo-dimeric complex

SupramoleculeName: homo-dimeric complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: PTS system glucose-specific EIICB component

MacromoleculeName: PTS system glucose-specific EIICB component / type: protein_or_peptide / ID: 1
Details: Extra residues at the C-terminus correspond to a 3C protease cleavage site
Number of copies: 2 / Enantiomer: LEVO
EC number: protein-Npi-phosphohistidine-D-glucose phosphotransferase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 51.691352 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFKNAFANLQ KVGKSLMLPV SVLPIAGILL GVGSANFSWL PAVVSHVMAE AGGSVFANMP LIFAIGVALG FTNNDGVSAL AAVVAYGIM VKTMAVVAPL VLHLPAEEIA SKHLADTGVL GGIISGAIAA YMFNRFYRIK LPEYLGFFAG KRFVPIISGL A AIFTGVVL ...String:
MFKNAFANLQ KVGKSLMLPV SVLPIAGILL GVGSANFSWL PAVVSHVMAE AGGSVFANMP LIFAIGVALG FTNNDGVSAL AAVVAYGIM VKTMAVVAPL VLHLPAEEIA SKHLADTGVL GGIISGAIAA YMFNRFYRIK LPEYLGFFAG KRFVPIISGL A AIFTGVVL SFIWPPIGSA IQTFSQWAAY QNPVVAFGIY GFIERCLVPF GLHHIWNVPF QMQIGEYTNA AGQVFHGDIP RY MAGDPTA GKLSGGFLFK MYGLPAAAIA IWHSAKPENR AKVGGIMISA ALTSFLTGIT EPIEFSFMFV APILYIIHAI LAG LAFPIC ILLGMRDGTS FSHGLIDFIV LSGNSSKLWL FPIVGIGYAI VYYTIFRVLI KALDLKTPGR EDATEDAKAT GTSE MAPAL VAAFGGKENI TNLDACITRL RVSVADVSKV DQAGLKKLGA AGVVVAGSGV QAIFGTKSDN LKTEMDEYIR NHLEL EVLF Q

UniProtKB: PTS system glucose-specific EIICB component

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Macromolecule #2: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 2 / Number of copies: 4 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6.2 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMHEPES-NaOH
150.0 mMNaCl
5.0 mMb-ME
0.03 mg/mLDDM
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 2.5 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 9036 / Average exposure time: 3.0 sec. / Average electron dose: 35.04 e/Å2
Details: Images were collected in the .eer format, split in 860 fractions
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6831987
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.0) / Number images used: 563331
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8qsr


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
Detailsinitial rigid body fitting in ChimeraX, flexible fitting in Isolde and refinement in Coot and Phenix
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9hnp:
Cryo-EM structure of the glucose-specific PTS transporter IICB from E. coli in an intermediate state

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